Processes for Preparing a Polypeptide

ABSTRACT

The present invention relates to an improved process for preparing a polypeptide or pharmaceutically acceptable salt thereof comprising L-tyrosine, L-alanine, L-glutamate, and L-lysine. The polypeptide or pharmaceutically acceptable salt thereof is preferably glatiramer acetate. The process comprises: (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride of a protected L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide; (b) admixing an acid with the protected polypeptide formed in Step (a) and a solvent, to form a product; and (c) admixing a substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate, and mixtures thereof, with the product formed in Step (b), and a solvent or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.

FIELD OF THE INVENTION

The present invention provides processes for preparing a polypeptide orpharmaceutically acceptable salt thereof. More specifically, theinvention provides processes for preparing glatiramer acetate.

BACKGROUND OF THE INVENTION

COPAXONE® is the trade name of glatiramer acetate, an FDA approved drugfor the treatment of multiple sclerosis. COPAXONE® is also known asCopolymer-1. The COPAXONE® label discloses that COPAXONE® consists ofthe acetate salts of synthetic polypeptides, containing fournaturally-occurring amino acids: L-glutamic acid, L-alanine, L-tyrosineand L-lysine with an average molar fraction of 0.141, 0.427, 0.095 and0.338, respectively, and has a weight average molecular weight of4.7-11.0 kilodaltons (kDa). COPAXONE® comprises a mixture ofpolypeptides having different molecular weights and sequences. Thestructural formula of COPAXONE® is:

(Glu, Ala, Lys, Tyr)_(x).xCH₃COOH

(C₅H₉NO₄.C₃H₇NO₂.C₆H₁₄N₂O₂.C₉H₁₁NO₃)_(x).xC₂H₄O₂

COPAXONE® is a white to off-white, sterile, lyophilized powdercontaining 20 mg glatiramer acetate and 40 mg of mannitol. It issupplied in single use vials for subcutaneous administration afterreconstitution with sterile water.

Processes for preparing Copolymer-1 or glatiramer acetate have beendescribed in U.S. Pat. Nos. 3,849,550; 5,800,808; 5,981,589; 6,048,898;6,054,430; 6,342,476; and 6,362,161. The process for the synthesis ofglatiramer acetate is based on the polymerization of N-carboxyanhydridesof tyrosine, alanine, γ-benzyl glutamate and N^(ε)-trifluoroacetyllysine in anhydrous dioxane at room temperature using diethylamine asinitiator, to form a protected polypeptide. The deblocking of theγ-benzyl groups (first deprotection) is accomplished by stirring theprotected polypeptide in hydrogen bromide/acetic acid at roomtemperature. These conditions also facilitate the cleavage of thecopolymer. The next step is the removal of the N^(ε)-trifluoroacetylgroups (second deprotection) of the copolymer by treatment with 1 Mpiperidine. In the final steps, glatiramer acetate is obtained bypurification of the copolymer through dialysis, followed by treatmentwith acetic acid to form the acetate salt and by another purification bydialysis against water. Thus, these prior art processes involve thepolymerization of four N-carboxyanhydrides, two deprotection steps, twopurification steps and one acetate salt formation step.

U.S. Pat. No. 6,620,847 describes a process for preparing Copolymer-1which involves treating trifluoroacetyl Copolymer-1 with aqueouspiperidine to form a solution of Copolymer-1 and purifying Copolymer-1.

U.S. Patent Application Publication No. 2004/0091956 describes athree-step process for preparing glatiramer acetate. The processinvolves polymerization of a mixture of the N-carboxyanhydrides ofL-alanine, L-tyrosine, protected L-glutamate and protected L-lysine, toobtain a protected polypeptide or salt thereof; and deprotection of theprotected polypeptide or salt thereof by either palladium catalytictransfer hydrogenation or palladium catalytic hydrogenation underhydrogen pressure.

SUMMARY OF THE INVENTION

The invention provides a process for preparing a polypeptide comprisingL-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceuticallyacceptable salt thereof, wherein said process comprises:

-   -   (i) polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine; N-carboxyanhydride of protected        L-glutamate; and N-carboxyanhydride of N-t-butoxycarbonyl        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide, wherein the        protected L-glutamate is selected from the group consisting of        γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures        thereof; and    -   (ii) adding an acid to the protected polypeptide formed in        Step (i) to form a polypeptide or a pharmaceutically acceptable        salt thereof, wherein said acid cleaves the γ-p-methoxybenzyl        group from the glutamate moiety and the N-t-butoxycarbonyl group        from the lysine moiety.

The invention provides a process for preparing a polypeptide comprisingL-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceuticallyacceptable salt thereof, wherein said process comprises treating aprotected polypeptide with an aqueous solution of an alkali or alkalineearth metal hydroxide to form a polypeptide or a pharmaceuticallyacceptable salt thereof.

The invention provides a process for preparing a polypeptide comprisingL-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceuticallyacceptable salt thereof, wherein said process comprises:

-   -   (a)² polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of a        protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide;    -   (b)² admixing an acid with the protected polypeptide formed in        Step (a)² and a solvent, to form a product; and    -   (c)² admixing a substance selected from the group consisting of        diisopropylamine, isopropylamine, ammonia and mixtures thereof,        with the product formed in Step (b)², and water or a mixture of        a solvent and water, to form a deprotected polypeptide or a        pharmaceutically acceptable salt thereof.

The invention provides a process for preparing a polypeptide comprisingL-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceuticallyacceptable salt thereof, wherein said process comprises:

-   -   (a)³ polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of a        protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide;    -   (b)³ admixing an acid with the protected polypeptide formed in        Step (a)³ and a solvent, to form a product; and    -   (c)³ admixing a substance selected from the group consisting of        an alkali or alkaline earth metal hydroxide, a carbonate, a        hydrogencarbonate and mixtures thereof, with the product formed        in Step (b)³, and a solvent or a mixture of a solvent and water,        to form a deprotected polypeptide or a pharmaceutically        acceptable salt thereof.

DESCRIPTION OF THE INVENTION

The present invention relates to processes for preparing a polypeptideor pharmaceutically acceptable salt thereof comprising L-tyrosine,L-alanine, L-glutamate and L-lysine. The polypeptide or pharmaceuticallyacceptable salt thereof is preferably glatiramer acetate.

I. Acid Hydrolysis Process.

In one embodiment of the invention, the process comprises:

-   -   (i) polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of protected        L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide, wherein the        protected L-glutamate is selected from the group consisting of        γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures        thereof; and    -   (ii) adding an acid to the protected polypeptide formed in        Step (i) to form a polypeptide or a pharmaceutically acceptable        salt thereof, wherein said acid cleaves the γ-p-methoxybenzyl        group from the glutamate moiety and the N-t-butoxycarbonyl group        from the lysine moiety.

In one embodiment of the invention, the process comprises:

-   -   (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of protected        L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected glatiramer, wherein the protected        L-glutamate is selected from the group consisting of        γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate, and        mixtures thereof;    -   (b) adding an acid to the protected glatiramer formed in        Step (a) to form a glatiramer, wherein said acid cleaves the        γ-p-methoxybenzyl group from the glutamate moiety and the        N-t-butoxycarbonyl group from the lysine moiety; and    -   (c) treating the glatiramer formed in Step (b) with acetic acid        to form glatiramer acetate.

In the polymerizing step of the processes of the invention, Step (i),Step (a), Step (a)¹, Step (a)¹′, Step (a)², Step (a)²′, Step (a)³ andStep (a)³′, the mixture of N-carboxyanhydride of L-tyrosine,N-carboxyanhydride of L-alanine, N-carboxyanhydride of protectedL-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, arepreferably polymerized at a temperature of from about 10° C. to about40° C., more preferably about 20° C. to about 30° C. The polymerizationreaction preferably takes place for a period of from about 2 hours toabout 80 hours, more preferably from about 20 hours to about 50 hours.Most preferably, the polymerization reaction takes place for a period ofabout 24 hours at a temperature of about 25° C.

The polar aprotic solvent is preferably selected from tetrahydrofuran,ethyl acetate, dimethyl furan, dimethylformamide, dioxane,dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide anddichloromethane. Most preferably, the polar aprotic solvent is1,4-dioxane. A mixture of polar aprotic solvents may also be used.

The initiator used in Step (i), Step (a), Step (a)¹, Step (a)¹′, Step(a)², Step (a)²′, Step (a)³ and Step (a)³′, of the processes of theinvention may be any alkylamine initiator, such as a dialkyl or atrialkylamine. Each of the alkyl groups preferably has 1-6 carbon atoms.A preferred alkylamine initiator is diethylamine. Preferably, thediethylamine is present in an amount of from about 0.001 weight percent(wt. %) to about 2 wt. %, more preferably from about 0.01 wt. % to about0.02 wt. %, based on the weight of the mixture of N-carboxyanhydride ofL-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride ofprotected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonylL-lysine.

In one embodiment of the invention, water is added to the polymerizationmixture following polymerization. The addition of water results inprecipitation of the protected polypeptide. The water is preferablyremoved from the mixture containing water and protected polypeptide byvacuum filtration and the recovered protected polypeptide is dried.Methods of drying are known to those skilled in the art, such as vacuumdrying.

In the deprotecting step, Step (ii), of the process of the invention, anacid is added to the protected polypeptide which is formed in Step (i)to form a polypeptide or a pharmaceutically acceptable salt thereof. Theacid cleaves the γ-p-methoxybenzyl group or γ-benzyl group from theglutamate moiety and the N-t-butoxycarbonyl group from the lysinemoiety. In addition, the acid cleaves the amide bonds of the polypeptideor pharmaceutically acceptable salt thereof forming heterogenouspolypeptide fragments.

In the deprotecting step, Step (b), of the process of the invention, anacid is added to the protected glatiramer formed in Step (a) to form aglatiramer. The acid cleaves the γ-p-methoxybenzyl group or the γ-benzylgroup from the glutamate moiety and the N-t-butoxycarbonyl group fromthe lysine moiety. In addition, the acid cleaves the amide bonds of theglatiramer forming heterogenous glatiramer fragments.

Suitable acids include, but are not limited to, acetic acid,hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane sulfonicacid, trifluoromethane sulfonic acid, phosphoric acid, trifluroaceticacid and sulfuric acid. A mixture of acids may also be used. Preferredacids are selected from trifluroacetic acid, a mixture of acetic acidand hydrochloric acid, a mixture of acetic acid and hydrogen bromide anda mixture of acetic acid and sulfuric acid. The acid may be added in theform of an aqueous solution.

The acid is preferably present in an amount of from about 0.1 wt. % toabout 100 wt. %, more preferably from about 1 wt. % to about 10 wt. %,based on the weight of the protected polypeptide or protectedglatiramer. Most preferably, the acid is present in an amount of fromabout 2 wt. % to about 6 wt. %, based on the weight of the protectedpolypeptide or protected glatiramer.

The temperature of the reaction medium during addition of the acid ispreferably from about 10° C. to about 40° C., more preferably 15° C. toabout 30° C. The acid is preferably added over a period of time fromabout 1 hour to about 30 hours, with stirring. Most preferably, the acidis added to the protected polypeptide or protected glatiramer at atemperature of about 25° C. for a period of from about 1 hour to about 8hours, with stirring.

Excess acid is preferably removed from the reaction mixture by purgingthe reaction mixture with nitrogen, lyophilization, or by means of arotary evaporator under vacuum to obtain a deprotected polypeptide insolid form. However, other separation techniques known to those skilledin the art may also be used.

The deprotected polypeptide or deprotected glatiramer in the form of afree base or acid addition salt is preferably dissolved in water or anaqueous acetic acid solution. Undesired low molecular weight polypeptideor glatiramer fragments, i.e., less than about 2 kDa, and high molecularweight polypeptide or glatiramer fragments, i.e., greater than about 40kDa, are preferably removed by such methods as dialysis ordiafiltration. Preferred membranes include Visking partially permeablecellulose membranes, such as a Size 6 membrane having a molecular weightcut-off of 12-14 kDa, available from Medicell International Ltd., andtangential flow filtration (TFF) membranes, such as a Pellicon XL PLCCC10 (50 cm²) or PLCCC 5 (50 cm²), available from Millipore. In apreferred embodiment of the invention, the deprotected polypeptide ordeprotected glatiramer is dissolved in water and subjected to dialysis,followed by dialysis in aqueous acetic acid solution.

The present inventors have determined that the desired molecular weightpolypeptide or a pharmaceutically acceptable salt thereof may becontrolled by dilution, concentration of the acid added in Step (ii) orStep (b), and/or time.

In one embodiment of the invention, water is removed from thedeprotected polypeptide. A preferred method of removal islyophilization. In lyophilization, the solution is frozen and placedunder vacuum so that the water (ice) vaporizes in the vacuum (sublimes)without melting and the non-water components (deprotected polypeptideand residual salt) are left behind in an undamaged state, i.e., withoutchemical decomposition. The dried product of lyophilization contains thedeprotected polypeptide and residual salt.

In one embodiment of the invention, the deprotected polypeptide istreated with glacial acetic acid to form glatiramer acetate salt. Theglatiramer acetate salt is collected preferably by lyophilization toyield a glatiramer acetate salt product.

II. Phase Transfer Process.

In one embodiment of the invention, the process for preparing apolypeptide comprising L-tyrosine, L-alanine, L-glutamate and L-lysine,or a pharmaceutically acceptable salt thereof, comprises treating aprotected polypeptide with an aqueous solution of an alkali or alkalineearth metal hydroxide to form a polypeptide or a pharmaceuticallyacceptable salt thereof.

In one embodiment of the invention, the process comprises:

-   -   (a)¹ polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of a        protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide; and    -   (b)¹ adding an aqueous solution of an alkali or alkaline earth        metal hydroxide to the protected polypeptide formed in Step (a)¹        to form a polypeptide or a pharmaceutically acceptable salt        thereof.

In one embodiment of the invention, the process comprises:

-   -   (a)^(1′) polymerizing a mixture of N-carboxyanhydride of        L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride        of a γ-benzyl L-glutamate L-glutamate and N-carboxyanhydride of        N^(ε)-trifluoroacetyl L-lysine, in a polar aprotic solvent in        the presence of an initiator, to form a protected glatiramer;    -   (b)¹′ adding an aqueous solution of an alkali or alkaline earth        metal hydroxide to the protected glatiramer formed in Step (a)¹′        to form a glatiramer; and    -   (c)¹′ treating the glatiramer with acetic acid to form        glatiramer acetate.

In the deprotecting steps, Step (b)¹ and Step (b)¹′, of the process ofthe invention, an aqueous solution of an alkali or alkaline earth metalhydroxide is added to the protected polypeptide formed in Step (a)¹ orto the protected glatiramer formed in Step (a)^(1′). In Step (b)¹ andStep (b)¹′, the protecting groups on the glutamic acid moiety, i.e., theγ-benzyl group, and the protecting groups on the lysine moiety, i.e.,N^(ε)-trifluoroacetyl group, are removed. In addition, the alkali oralkaline earth metal hydroxide cleaves the amide bonds of thepolypeptide or glatiramer forming heterogenous polypeptide or glatiramerfragments.

While not wishing to be bound by any particular theory, the presentinventors believe that the removal of the protecting groups from theprotected polypeptide or protected glatiramer results in a phasetransfer of the deprotected polypeptide or deprotected glatiramer fromthe organic phase to the aqueous phase.

In the absence of a buffer in Step (b)¹ or Step (b)¹′, the pH duringStep (b)¹ and Step (b)¹′, is generally about 13 to about 14 after theaddition of the aqueous solution of an alkali or alkaline earth metalhydroxide. In the presence of a buffer in Step (b)¹ or Step (b)^(b)′,the pH is generally about 8 to about 12. The buffer may be added orformed in situ. A preferred buffer is an acetate buffer such as sodiumacetate.

The alkali or alkaline earth metal hydroxide is preferably selected fromcalcium hydroxide, lithium hydroxide, magnesium hydroxide, potassiumhydroxide and sodium hydroxide. More preferably, the alkali or alkalineearth metal hydroxide is sodium hydroxide. A combination of alkali oralkaline earth metal hydroxides may also be used.

The alkali or alkaline earth metal hydroxide is preferably present in anamount of from about 0.1 wt. % to about 400 wt. %, more preferably fromabout 10 wt. % to about 300 wt. %, based on the weight of the protectedpolypeptide or protected glatiramer. Most preferably, the alkali oralkaline earth metal hydroxide is present in an amount of from about 140wt. % to about 260 wt. %, based on the weight of the protectedpolypeptide or protected glatiramer.

An aqueous solution of an alkali or alkaline earth metal hydroxide isadded to the protected polypeptide or protected glatiramer preferably ata temperature of from about −78° C. to about 40° C., more preferably−25° C. to about 30° C., for a period of time preferably from about 1hour to about 30 hours. Most preferably, an aqueous solution of analkali or alkaline earth metal hydroxide is added to the protectedpolypeptide or protected glatiramer at a temperature from about −10° C.to about 10° C., e.g., 0° C., for a period of from about 1 hour to about8 hours, with stirring. The addition of an aqueous solution of an alkalior alkaline earth metal hydroxide results in a phase separation whereinan organic phase and an aqueous phase are formed.

The organic phase substantially contains the polar aprotic solvent andthe protected polypeptide or protected glatiramer. The aqueous phasesubstantially contains water, the alkali or alkaline earth metalhydroxide, and the deprotected polypeptide or deprotected glatiramer inthe form of a free base. The aqueous phase and the organic phase arepreferably separated by using a centrifuge and decanting the organicphase.

An additional advantage of the process of the invention is that theaddition of the alkali or alkaline earth metal hydroxide causesdeprotection of the protected polypeptide to form a deprotectedpolypeptide, or deprotection of the protected glatiramer to form adeprotected glatiramer. In addition, the alkali or alkaline earth metalhydroxide causes cleavage of the amide bonds in the deprotectedpolypeptide or deprotected glatiramer to form polypeptide or glatiramerfragments.

The aqueous phase is preferably treated with an organic or mineral acidto achieve a pH of about 7 to about 8. Such organic or mineral acids toadjust the pH are well-known to those skilled in the art and include,but are not limited to, acetic acid, formic acid, oxalic acid andhydrochloric acid. Dilute hydrochloric acid is preferred. The amount oforganic or mineral acid used is preferably an equivalent amount based onthe amount of the alkali or alkaline earth metal hydroxide added duringStep (b)¹ or Step (b)¹′ which is sufficient to produce a pH of about 7to about 8.

Undesired low molecular weight polypeptide or glatiramer fragments,i.e., less than about 2 kDa, and high molecular weight polypeptide orglatiramer fragments, i.e., greater than about 40 kDa, are preferablyremoved by such methods as dialysis or diafiltration. Preferredmembranes include Visking partially permeable cellulose membranes suchas a Size 6 membrane having a molecular weight cut-off of 12-14 kDa,available from Medicell International Ltd., and tangential flowfiltration (TFF) membranes, such as a Pellicon XL PLCCC 10 (50 cm²) orPLCCC 5 (50 cm²), available from Millipore. In a preferred embodiment ofthe invention, the deprotected polypeptide or deprotected glatiramer issubjected to dialysis in water, followed by dialysis in aqueous aceticacid solution.

III. Process Using an Amine or Ammonia.

In one embodiment of the invention, the process comprises:

-   -   (a)² polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of a        protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide;    -   (b)² admixing an acid with the protected polypeptide formed in        Step (a)² and a solvent, to form a product, preferably, the acid        is admixed with a solution or suspension comprising the        protected polypeptide and solvent; and    -   (c)² admixing a substance selected from the group consisting of        diisopropylamine, isopropylamine, ammonia, and mixtures thereof,        with the product formed in Step (b)², and water or a mixture of        a solvent and water, to form a deprotected polypeptide or a        pharmaceutically acceptable salt thereof.

In one embodiment of the invention, the process comprises:

-   -   (a)^(2′) polymerizing a mixture of N-carboxyanhydride of        L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride        of a protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected glatiramer, wherein said        protected L-glutamate is selected from the group consisting of        γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures        thereof;    -   (b)^(2′) admixing an acid with the protected glatiramer formed        in Step (a)^(2′) and a solvent, to form a product;    -   (c)^(2′) admixing a substance selected from the group consisting        of diisopropylamine, isopropylamine, ammonia and mixtures        thereof, with the product formed in Step (b)^(2′), and water or        a mixture of a solvent and water, to form a deprotected        glatiramer; and    -   (d)²′ treating the deprotected glatiramer formed in Step        (c)^(2′) with acetic acid to form glatiramer acetate.

In the first deprotecting step, Step (b)² and Step (b)²′ of the processof the invention, an acid is admixed with the protected polypeptideformed in Step (a)² or protected glatiramer formed in Step (a)²′, and asolvent to form a product. Preferably, the acid is added to a mixture,i.e., a solution or suspension, comprising the protected polypeptide orprotected glatiramer and a solvent. In Step (b)² and Step (b)²′ theprotecting groups on the glutamic acid moiety, i.e., γ-p-methoxybenzylgroup and/or γ-benzyl group, are removed. While not wishing to be boundby any particular theory, the present inventors believe that the acidalso cleaves amide bonds of the protected polypeptide or protectedglatiramer forming heterogenous polypeptide or glatiramer fragments.

Suitable acids include, but are not limited to, acetic acid,hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane sulfonicacid, trifluoromethane sulfonic acid, phosphoric acid, trifluroaceticacid and sulfuric acid. A mixture of acids may also be used. Preferredacids are selected from trifluroacetic acid, a mixture of acetic acidand hydrochloric acid, a mixture of acetic acid and hydrogen bromide anda mixture of acetic acid and sulfuric acid. The acid may be added in theform of an aqueous solution.

The acid is preferably present in an amount of from about 0.1 wt. % toabout 100 wt. %, more preferably from about 1 wt. % to about 10 wt. %,based on the weight of the protected polypeptide or protectedglatiramer. Most preferably, the acid is present in an amount of fromabout 2 wt. % to about 6 wt. %, based on the weight of the protectedpolypeptide or protected glatiramer.

The temperature of the reaction medium during addition of the acid ispreferably from about 10° C. to about 40° C., more preferably 15° C. toabout 30° C. The acid is preferably added over a period of time fromabout 1 hour to about 30 hours, with stirring. Most preferably, the acidis added to the protected polypeptide or protected glatiramer at atemperature of about 25° C. for a period of from about 1 hour to about 8hours, with stirring.

The solvent used in the first deprotection step, Step (b)² and Step(b)²′, is selected from polar protic solvents and polar aproticsolvents. Preferably, the solvent used in Step (b)² and Step (b)²′ isselected from acetic acid, tetrahydrofuran, ethyl acetate, dimethylfuran, dimethylformamide, 1,4-dioxane, dimethoxyethane,1,2-dichloroethylene, dimethylsulfoxide and dichloromethane. A mixtureof solvents may also be used. Most preferably, the solvent used in Step(b)² and Step (b)²′ is tetrahydrofuran or acetic acid.

The amount of solvent used in Step (b)² and Step (b)²′ is preferablyfrom about 1-fold (wt.) to about 1,000-fold (wt.), more preferably, fromabout 10-fold (wt.) to about 500-fold (wt.), based on the amount ofprotected polypeptide or protected glatiramer which is used in Step (b)²or Step (b)^(2′).

In the second deprotecting step, Step (c)² and Step (c)²′ of the processof the invention, a substance selected from the group consisting ofdiisopropylamine, isopropylamine, ammonia and mixtures thereof, isadmixed with the product formed in Step (b)² or Step (b)²′, and water ora mixture of a solvent and water, to form a deprotected polypeptide ordeprotected glatiramer.

Preferably, the substance selected from the group consisting ofdiisopropylamine, isopropylamine, ammonia and mixtures thereof, is addedto a mixture, i.e., a solution or suspension, comprising the productformed in Step (b)² or Step (b)²′, and water or a mixture of a solventand water. The ammonia is in the form of NH₃ (aqueous) or NH₃ (gas).Preferably an aqueous solution of ammonia is used having a pH of about 7to about 14. The addition of a substance selected from the groupconsisting of diisopropylamine, isopropylamine, ammonia and mixturesthereof, to the product formed in Step (b)² or Step (b)²′ preferablyremoves the protecting group, such as N^(ε)-trifluoroacetyl group, ofthe lysine moiety. Preferably the deprotected polypeptide is adeprotected glatiramer in the form of a free base.

The substance selected from the group consisting of diisopropylamine,isopropylamine, ammonia and mixtures thereof, is preferably present inan amount of from about 1-fold (wt.) to about 1,000-fold (wt), morepreferably from about 10-fold (wt.) to about 500-fold (wt), based on thetotal weight of the product of Step (b)² or Step (b)²′ which is used inStep (c)² or Step (c)²′. Most preferably, the substance selected fromthe group consisting of diisopropylamine, isopropylamine, ammonia andmixtures thereof, is present in an amount of from about 50-fold (wt.) toabout 150-fold (wt).

The substance selected from the group consisting of diisopropylamine,isopropylamine, ammonia and mixtures thereof, is preferably admixed witha solution or suspension comprising the product formed in Step (b)² orStep (b)²′, and water or a mixture of a solvent and water, at atemperature of from about 10° C. to about 60° C., more preferably 15° C.to about 40° C., for a period of time preferably from about 1 minute toabout 60 hours, to form a deprotected polypeptide or deprotectedglatiramer. Suitable solvents include, but are not limited to,tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide,1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxideand dichloromethane. A preferred solvent is tetrahydrofuran.

More preferably, the substance selected from the group consisting ofdiisopropylamine, isopropylamine, ammonia and mixtures thereof, isadmixed with a solution or suspension comprising the product formed inStep (b)² or Step (b)²′, and water or a mixture of a solvent and water,at a temperature of from about 20° C. to about 30° C., for a period offrom about 1 hour to about 30 hours, to form a deprotected polypeptideor deprotected glatiramer. Unreacted diisopropylamine or ammonia, or anysolvent or water, is preferably removed by evaporation or vacuumdistillation.

Undesired low molecular weight polypeptide or glatiramer fragments,i.e., less than about 2 kDa, and high molecular weight polypeptide orglatiramer fragments, i.e., greater than about 40 kDa, are preferablyremoved by such methods as dialysis or diafiltration. Preferredmembranes include Visking partially permeable cellulose membranes, suchas a Size 6 membrane having a molecular weight cut-off of 12-14 kDa,available from Medicell International Ltd., and TFF membranes, such as aPellicon XL PLCCC 10 (50 cm²) or PLCCC 5 (50 cm²), available fromMillipore. In a preferred embodiment of the invention, the deprotectedpolypeptide or deprotected glatiramer is subjected to dialysis in water,followed by dialysis in aqueous acetic acid solution.

IV. Process Using an Alkali or Alkaline Earth Metal Hydroxide, Carbonateor Hydrogencarbonate.

In one embodiment of the invention, the process comprises:

-   -   (a)³ polymerizing a mixture of N-carboxyanhydride of L-tyrosine,        N-carboxyanhydride of L-alanine, N-carboxyanhydride of a        protected L-glutamate and N-carboxyanhydride of a protected        L-lysine, in a polar aprotic solvent in the presence of an        initiator, to form a protected polypeptide;    -   (b)³ admixing an acid with the protected polypeptide formed in        Step (a)³ and a solvent, to form a product, preferably, the acid        is admixed with a solution or suspension comprising the        protected polypeptide and solvent; and    -   (c)³ admixing a substance selected from the group consisting of        an alkali or alkaline earth metal hydroxide, a carbonate, a        hydrogencarbonate and mixtures thereof, with the product formed        in Step (b)³, and a solvent or a mixture of a solvent and water,        to form a deprotected polypeptide or a pharmaceutically        acceptable salt thereof.

In one embodiment of the invention, the process comprises:

-   -   (a)³′ polymerizing a mixture of N-carboxyanhydride of        L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride        of a protected L-glutamate and N-carboxyanhydride of        N^(ε)-trifluoroacetyl L-lysine, in a polar aprotic solvent in        the presence of an initiator, to form a protected glatiramer,        wherein said protected L-glutamate is selected from the group        consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl        L-glutamate and mixtures thereof;    -   (b)³′ admixing an acid with a mixture comprising the protected        glatiramer formed in Step (a)³ and a solvent, to form a product;    -   (c)³′ admixing a substance selected from the group consisting of        an alkali or alkaline earth metal hydroxide, a carbonate, a        hydrogencarbonate and mixtures thereof, with a mixture        comprising the product formed in Step (b)³′, and a solvent or a        mixture of a solvent and water, to form a deprotected        glatiramer; and    -   (d)^(3′) treating the deprotected glatiramer formed in Step        (c)³′ with acetic acid to form glatiramer acetate.

In the first deprotecting step, Step (b)³ and Step (b)³′ of the processof the invention, an acid is admixed with the protected polypeptideformed in Step (a)³ or protected glatiramer formed in Step (a)³′, and asolvent to form a product. Preferably, the acid is added to a mixture,i.e., a solution or suspension, comprising the protected polypeptide orprotected glatiramer and a solvent. In Step (b)³ and Step (b)³′ theprotecting groups on the glutamic acid moiety, i.e., γ-p-methoxybenzylgroup and/or γ-benzyl group, are removed. While not wishing to be boundby any particular theory, the present inventors believe that the acidalso cleaves amide bonds of the protected polypeptide or protectedglatiramer forming heterogenous polypeptide fragments.

Suitable acids include, but are not limited to, acetic acid,hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane sulfonicacid, trifluoromethane sulfonic acid, phosphoric acid, trifluroaceticacid and sulfuric acid. A mixture of acids may also be used. Preferredacids are selected from trifluroacetic acid, a mixture of acetic acidand hydrochloric acid, a mixture of acetic acid and hydrogen bromide anda mixture of acetic acid and sulfuric acid. The acid may be added in theform of an aqueous solution.

The acid is preferably present in an amount of from about 0.1 wt. % toabout 100 wt. %, more preferably from about 1 wt. % to about 10 wt. %,based on the weight of the protected polypeptide or protectedglatiramer. Most preferably, the acid is present in an amount of fromabout 2 wt. % to about 6 wt. %, based on the weight of the protectedpolypeptide or protected glatiramer.

The temperature of the reaction medium during addition of the acid ispreferably from about 10° C. to about 40° C., more preferably 15° C. toabout 30° C. The acid is preferably added over a period of time fromabout 1 hour to about 30 hours, with stirring. Most preferably, the acidis added to the protected polypeptide or protected glatiramer at atemperature of about 25° C. for a period of from about 1 hour to about 8hours, with stirring.

The solvent used in the first deprotection step, Step (b)³ and Step(b)³′, is selected from polar protic solvents and polar aproticsolvents. Preferably, the solvent used in Step (b)³ and Step (b)³′ isselected from acetic acid, tetrahydrofuran, ethyl acetate, dimethylfuran, dimethylformamide, 1,4-dioxane, dimethoxyethane,1,2-dichloroethylene, dimethylsulfoxide and dichloromethane. A mixtureof solvents may also be used. Most preferably, the solvent used in Step(b)² and Step (b)²′ is tetrahydrofuran or acetic acid.

The amount of solvent used in Step (b)³ and Step (b)³′ is preferablyfrom about 1-fold (wt.) to about 1,000-fold (wt.), more preferably, fromabout 10-fold (wt.) to about 500-fold (wt.), based on the amount ofprotected polypeptide or protected glatiramer which is used in Step (b)³or Step (b)^(3′).

In the second deprotecting step, Step (c)³ and Step (c)³′, of theprocess of the invention, a substance selected from the group consistingof an alkali or alkaline earth metal hydroxide, a carbonate, ahydrogencarbonate and mixtures thereof, is admixed with the productformed in Step (b)³ or Step (b)³′, and water or a mixture of a solventand water, to form a deprotected polypeptide or deprotected glatiramer.

Preferably, the substance selected from the group consisting of analkali or alkaline earth metal hydroxide, a carbonate, ahydrogencarbonate and mixtures thereof, is added to a mixture, i.e., asolution or suspension, comprising the product formed in Step (b)³ orStep (b)³′, and water or a mixture of a solvent and water. The additionof a substance selected from the group consisting of an alkali oralkaline earth metal hydroxide, a carbonate, a hydrogencarbonate andmixtures thereof, to the product formed in Step (b)³ or Step (b)³′preferably removes the N^(ε)-trifluoroacetyl group of the lysine moiety.Preferably the deprotected polypeptide is a deprotected glatiramer inthe form of a free base.

The substance selected from the group consisting of an alkali oralkaline earth metal hydroxide, a carbonate and a hydrogencarbonate, andincludes calcium hydroxide, lithium hydroxide, magnesium hydroxide,potassium hydroxide, barium hydroxide, sodium hydroxide, calciumcarbonate, lithium carbonate, magnesium carbonate, potassium carbonate,sodium carbonate, calcium hydrogencarbonate, lithium hydrogencarbonate,magnesium hydrogencarbonate, potassium hydrogencarbonate and sodiumhydrogencarbonate. More preferably, the substance used in Step (c)³ orStep (c)³′ is selected from the group consisting of sodium hydroxide,lithium hydroxide and potassium hydroxide.

The substance selected from the group consisting of an alkali oralkaline earth metal hydroxide, a carbonate, a hydrogencarbonate andmixtures thereof, is preferably present in an amount of from about1-fold (wt.) to about 1,000-fold (wt), more preferably from about10-fold (wt.) to about 500-fold (wt), based on the total weight of theproduct of Step (b)³ or Step (b)³′ which is used in Step (c)³ or Step(c)³′. Most preferably, the substance selected from the group consistingof an alkali or alkaline earth metal hydroxide, a carbonate, ahydrogencarbonate and mixtures thereof, is present in an amount of fromabout 50-fold (wt.) to about 150-fold (wt).

The substance selected from the group consisting of an alkali oralkaline earth metal hydroxide, a carbonate, a hydrogencarbonate andmixtures thereof, is preferably admixed with a solution or suspensioncomprising the product formed in Step (b)³ or Step (b)³′, and water or amixture of a solvent and water, at a temperature of from about 10° C. toabout 60° C., more preferably 15° C. to about 40° C., for a period oftime preferably from about 1 minute to about 60 hours, to form adeprotected polypeptide or deprotected glatiramer. Suitable solventsinclude, but are not limited to, tetrahydrofuran, ethyl acetate,dimethyl furan, dimethylformamide, 1,4-dioxane, dimethoxyethane,1,2-dichloroethylene, dimethylsulfoxide and dichloromethane. A preferredsolvent is tetrahydrofuran.

More preferably, the substance selected from the group consisting of analkali or alkaline earth metal hydroxide, a carbonate, ahydrogencarbonate and mixtures thereof, is admixed with a solution orsuspension comprising the product formed in Step (b)³ or Step (b)³′, andwater or a mixture of a solvent and water, at a temperature of fromabout 20° C. to about 30° C., for a period of from about 1 hour to about30 hours, to form a deprotected polypeptide or deprotected glatiramer.Unreacted alkali or alkaline earth metal hydroxide, carbonate,hydrogencarbonate or any solvent or water, is preferably removed byevaporation or vacuum distillation.

The amount of solvent or a mixture of a solvent and water which is usedin Step (c)³ or Step (c)³′ is preferably from about 1-fold (wt.) toabout 1,000-fold (wt.), more preferably, from about 10-fold (wt.) toabout 500-fold (wt.), based on the total weight of the product of Step(b)³ or Step (b)³′ which is used in Step (c)³ or Step (c)³′.

After the second deprotection step, Step (c)³ and Step (c)³′, any layersare separated preferably in separatory funnel, or the solvent ispreferably removed by evaporation or vacuum distillation. Thedeprotected polypeptide or deprotected glatiramer is preferably obtainedas a solution in water.

Undesired low molecular weight polypeptide or glatiramer fragments,i.e., less than about 2 kDa, and high molecular weight polypeptide orglatiramer fragments, i.e., greater than about 40 kDa, are preferablyremoved by such methods as dialysis or diafiltration. Preferredmembranes include Visking partially permeable cellulose membranes, suchas a Size 6 membrane having a molecular weight cut-off of 12-14 kDa,available from Medicell International Ltd., and TFF membranes, such as aPellicon XL PLCCC 10 (50 cm²) or PLCCC 5 (50 cm²), available fromMillipore. In a preferred embodiment of the invention, the deprotectedpolypeptide or deprotected glatiramer is subjected to dialysis in water,followed by dialysis in aqueous acetic acid solution.

The weight average molecular weight of the polypeptide or apharmaceutically acceptable salt thereof which is prepared in accordancewith the processes of the invention is preferably from about 2 kDa toabout 30 kDa, more preferably from about 4.7 Da to about 11 kDa, andmost preferably from about 7 kDa to about 10 kDa, as determined by gaspermeation chromatography (GPC). Preferably, the polypeptide orpharmaceutically acceptable salt thereof is substantially free ofpolypeptide fragments having a molecular weight of greater than about 40kDa. Preferably, the polypeptide or pharmaceutically acceptable saltthereof is substantially free of polypeptide fragments having amolecular weight of less than about 2 kDa. As used herein,“substantially free” means less than about 5% by weight, more preferablyless than about 2.5% by weight of the polypeptide prepared according tothe process of the invention.

The polypeptide or pharmaceutically acceptable salt thereof, preferablyglatiramer acetate, prepared according to the process of the inventionmay be formulated by conventional methods known in the art.

In one embodiment of the invention, the polypeptide or pharmaceuticallyacceptable salt thereof, which is prepared by the process of theinvention, is dissolved or suspended in an acceptable pharmaceuticalliquid vehicle, such as water, and the solution or suspension isinjected into the body.

In one embodiment of the invention, the glatiramer acetate salt isdissolved in a mixture containing water and mannitol, and the solutionis injected into the body.

Typically, the polypeptide or a pharmaceutically acceptable salt thereofis administered daily to patients suffering from multiple sclerosis,e.g., at a dosage of 20 mg of glatiramer acetate.

The following non-limiting examples illustrate further aspects of theinvention.

Examples 1-7 relate to an acid hydrolysis process for preparingglatiramer acetate.

EXAMPLE 1 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-alanine (860 mg, 7.5 mmol), N-carboxyanhydrideof γ-benzyl L-glutamate (600 mg, 2.3 mmol), N-carboxyanhydride ofN-t-butoxycarbonyl L-lysine (1410 mg, 5.2 mmol) and N-carboxyanhydrideof L-tyrosine (300 mg, 1.4 mmol) are placed in a three-neck flask.Distilled anhydrous dioxane (57 mL) is added. Diethylamine (3.4 μL) isadded. The resulting mixture is stirred mechanically for 24 hours at atemperature of approximately 22-25° C. The mixture is slowly poured into100 mL deionized water and filtered under vacuum. The solid is keptunder vacuum for 12 hours.

EXAMPLE 2 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-alanine (430 mg, 3.75 mmol), N-carboxyanhydrideof γ-benzyl L-glutamate (300 mg, 1.15 mmol), N-carboxyanhydride ofN-t-butoxycarbonyl L-lysine (705 mg, 2.6 mmol) and N-carboxyanhydride ofL-tyrosine (150 mg, 0.7 mmol) are placed in a three-neck flask.Distilled anhydrous dioxane (28.5 mL) is added. Diethylamine (1.7 μL) isadded. The resulting mixture is stirred mechanically for 24 hours at atemperature of approximately 22-25° C. The mixture is slowly poured into100 mL deionized water and filtered under vacuum. The solid is keptunder vacuum for 12 hours.

EXAMPLE 3

Deprotecting the Protected Polypeptide Prepared in Example 1 withHBr/Acetic Acid.

The protected polypeptide prepared in Example 1 (200 mg) is suspended in7 mL of 33% hydrogen bromide in acetic acid. The starting materialslowly dissolves forming a red brown solution. The mixture is stirredfor 17 hours at a temperature of approximately 22° C. The solution ofHBr/acetic acid is evaporated to dryness using a rotary evaporator underreduced pressure. To this residue, 100 mL of water is added to dissolvethe solid. The solution is placed in a Visking partially permeablecellulose membrane which is in the form of a tube, Size 6, having amolecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter of27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is availablefrom Medicell International Ltd. The tube containing the solution isstirred in a beaker of water. Polypeptide fragments having a molecularweight less than about 2 kDa are removed by osmosis from the dialysistube. The tube is removed from the water and stirred in a beakercontaining 0.3% acetic acid in water. The resulting product is removedfrom the tube and lyophilized to obtain glatiramer acetate as a purewhite solid.

EXAMPLE 4

Deprotecting the Protected Polypeptide Prepared in Example 1 withHCl/Acetic Acid.

The protected polypeptide prepared in Example 1 (200 mg) is suspended in20 mL of a mixture prepared of 9.4 mL concentrated hydrochloric acidadjusted to 20 mL with glacial acetic acid. The starting material slowlydissolves forming a slightly turbid solution. The mixture is stirred for17 hours at a temperature of approximately 22° C. The solution ofHCl/acetic acid is evaporated to dryness using a rotary evaporator underreduced pressure. To this residue, 100 mL of water is added to dissolvethe solid. The solution is placed in a Visking partially permeablecellulose membrane which is in the form of a tube, Size 6, having amolecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter of27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is availablefrom Medicell International Ltd. The tube containing the solution isstirred in a beaker of water. Polypeptide fragments having a molecularweight less than about 2 kDa are removed by osmosis from the dialysistube. The tube is removed from the water and stirred in a beakercontaining 0.3% acetic acid in water. The resulting product is removedfrom the tube and lyophilized to obtain glatiramer acetate as a purewhite solid.

EXAMPLE 5

Deprotecting the Protected Polypeptide Prepared in Example 2 withHCl/Acetic Acid.

The protected polypeptide prepared in Example 2 (200 mg) is suspended in20 mL of a mixture prepared of 9.4 mL concentrated hydrochloric acidadjusted to 20 mL with glacial acetic acid. The starting material slowlydissolves forming a slightly turbid solution. The mixture is stirred for17 hours at a temperature of approximately 22° C. The solution ofHCl/acetic acid is evaporated to dryness using a rotary evaporator underreduced pressure. To this residue, 100 mL of water is added to dissolvethe solid. The solution is placed in a Visking partially permeablecellulose membrane which is in the form of a tube, Size 6, having amolecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter of27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is availablefrom Medicell International Ltd. The tube containing the solution isstirred in a beaker of water. Polypeptide fragments having a molecularweight less than about 2 kDa are removed by osmosis from the dialysistube. The tube is removed from the water and stirred in a beakercontaining 0.3% acetic acid in water. The resulting product is removedfrom the tube and lyophilized to obtain glatiramer acetate as a purewhite solid.

EXAMPLE 6 Relative Molecular Weight Determination by GPC Using a UVDetector. GPC-UV Detector Conditions:

Eluent: Phosphate buffer 0.05 M, pH 7.4, 5.6 g Na₂HPO₄, 116 g NaCl/4 Lwater Column: PSS Suprema, 10 μm, 100 A, 8 × 300 mm. Temperature: 23° C.Pump: TSP AS 3000 autosampler inj. - Vol.: 50 μL Concentration: about2.0 mg/mL Detector: TSP UV2000 at 276 nm GPC- PSS WinGPC Vers. 7.2Software: Samples: COPAXONE ® (Lot # 5308036) wherein the mannitol hasbeen removed by dialysis COPAXONE ® (Lot # 8040341) wherein the mannitolhas been removed by dialysis Sample A is glatiramer acetate prepared inExample 4 according to the process of the invention Sample B isglatiramer acetate prepared in Example 5 according to the process of theinvention Sample The weighted samples are dispersed in the eluent andPreparation: allowed to stand for full hydration at room temperature forabout 12 hours. The sample solution is filtered through a 1.0 filterunit (Schleicher & Schuell)

The test results for GPC are summarized in Table I.

TABLE I Sample M_(w) [kD] COPAXONE ® (Lot # 5308036) 7.923 COPAXONE ®(Lot # 8040341) 9.524 Sample A 8.551 Sample B 7.689

The test results in Table I clearly show that the weight averagemolecular weight of glatiramer acetate prepared by the process of theinvention, Samples A and B, is 8.551 kDa and 7.689 kDa, respectively,and the weight average molecular weight of COPAXONE® is 7.923 kDa and9.524 kDa, depending on the lot number.

EXAMPLE 7 Relative Molecular Weight Determination by Gel PermeationChromatography (GPC) Using a Refractive Index Detector. GPC-RI DetectorConditions:

Eluent: Phosphate buffer 0.05 M, pH 7.4, 5.6 g Na₂HPO₄, 116 g NaCl/4 Lwater Column: PSS Suprema, 10 μm, 100 A, 8 × 300 mm Temperature: 23° C.Pump: TSP AS 3000 autosampler inj. - Vol.: 50 μL Concentration: about2.0 mg/mL Detector: Shodex RI 71 GPC-Software: PSS WinGPC Vers. 7.2Samples: COPAXONE ® (Lot # 5308036) wherein the mannitol has beenremoved by dialysis COPAXONE ® (Lot # 8040341) wherein the mannitol hasbeen removed by dialysis Sample A is glatiramer acetate prepared inExample 4 according to the process of the invention Sample B isglatiramer acetate prepared in Example 5 according to the process of theinvention Sample The weighted samples are dispersed in the Preparation:eluent and allowed to stand for full hydration at room temperature forabout 12 hours. The sample solution is filtered through a 1.0 filterunit (Schleicher & Schuell)

The test results for GPC with RI detector are summarized in Table II.

TABLE II Sample M_(w) [kD] COPAXONE ® (Lot # 5308036) 8.663 COPAXONE ®(Lot # 8040341) 9.641 Sample A 9.581 Sample B 8.224

The test results in Table II clearly show that the weight averagemolecular weight of glatiramer acetate prepared by the process of theinvention, Samples A and B, is 9.581 kDa and 8.224 kDa, respectively,and the weight average molecular weight of COPAXONE® is 8.663 kDa and9.641 kDa, depending on the lot number.

Examples 8-17 relate to a phase transfer process for preparingglatiramer acetate.

The present inventors have established a scale of 1 (low) to 5 (high) toquantify the influence of the alkaline compound (sodium hydroxide) onglatiramer formation. The test results are summarized in Table III.

TABLE III Recovery of Desired NaOH Conc. Molecular Low Product with in15 mL Weight Product Molecular Desired THF + 75 mg from Aqueous WeightMolecular Glatiramer Free Phase Phase Polypeptide Weight Base SeparationSM = 75 mg Formation Cut-off 0.1 N/5 mL No 1 1 1 0.25 N/5 mL 1 5 1 50.25 N/10 mL 1 5 1 5 0.5 N/5 mL 3 3 2 4 0.5 N/10 mL 4 3 3 3 0.5 N/10mL + 5 3 2 4 10 mL water 1.0 N/5 mL 5 3 5 1

The results in Table III clearly show that 0.1-1.0 N sodium hydroxidesolutions may be used to prepare the polypeptide or a pharmaceuticallyacceptable salt thereof products of the invention. The results in TableIII also show that the use of a sodium hydroxide concentration of atleast 0.25 N is preferred to facilitate phase separation of the organicphase and aqueous phase.

EXAMPLE 8 Polymerization Step (a).

N-Carboxyanhydride of tyrosine (30 mg, 0.010 mm), N-carboxyanhydride ofalanine (62 mg, 0.054 mm), N-carboxyanhydride of γ-benzyl glutamate (42mg, 0.016 mm) and N-carboxyanhydride of E-N-trifluoroacetyllysine (100mg, 0.037 mm), are placed in a single-neck flask with a magneticstirrer. To this mixture is added 20 mL of dry tetrahydrofuran. A clearsolution is obtained. Diethylamine (10 μL) is added. The resultingmixture is stirred for 48 hours. The tetrahydrofuran is removed byevaporation. Water (150 mL) is added to the residue and the stirring iscontinued. A white solid is obtained. The solid is filtered and dried ina dessicator under vacuum.

The yield is determined to be 154 mg.

EXAMPLE 9 Deprotecting Step (b).

Protected glatiramer acetate, 75 mg, prepared in Example 8 istransferred to a single-neck flask provided with a magnetic stirrer.Tetrahydrofuran (15 mL) is added to the flask and stirred. A clearsolution is obtained. Aqueous solution of sodium hydroxide (8 mL) 0.5 N,is added. The addition of the sodium hydroxide solution results in themixture becoming hazy. The mixture is stirred for 1 hour at 24-26° C.The formation of two phases is observed. The bottom layer is separatedand acidified using dilute aqueous 1 N HCl solution to pH=6.0 withstirring. The crude glatiramer free base solution is filtered using anylon filter (0.2 micron Nylon Acrodisk).

EXAMPLE 10

Protected glatiramer acetate, 75 mg, prepared in Example 8 istransferred to a single-neck flask provided with a magnetic stirrer.Tetrahydrofuran (15 mL) is added to the flask and stirred. A clearsolution is obtained. Aqueous solution of sodium hydroxide (10 mL) 0.25N is added. The addition of the sodium hydroxide solution results in themixture becoming hazy. The mixture is stirred for 16 hours at 25-26° C.The reaction mixture is centrifuged for 15 minutes. The formation of twophases is observed. The bottom layer is separated and acidified usingdilute aqueous HCl solution to pH=7-7.5 with stirring. Stirring iscontinued for an additional 30 minutes and the pH is determined to bepH=8.0. The crude glatiramer free base solution is filtered.

EXAMPLE 11 Polymerization Step.

N-Carboxyanhydride of tyrosine (30 mg, 0.010 mm), N-carboxyanhydride ofalanine (62 mg, 0.054 mm), N-carboxyanhydride of γ-benzyl glutamate (42mg, 0.016 mm) and N-carboxyanhydride of E-N-trifluoroacetyllysine (100mg, 0.037 mm), are placed in a single-neck flask with a magneticstirrer. To this mixture is added 20 mL of dry dioxane. A clear solutionis obtained. Diethylamine (10 μL) is added. The resulting mixture isstirred for 48 hours. To this, water (150 mL) is added slowly withstirring. A white solid is obtained. The solid is filtered and dried ina dessicator under vacuum.

The yield is determined to be 170 mg.

EXAMPLE 12 Deprotecting Step.

Protected glatiramer acetate, 75 mg, prepared in Example 11 istransferred to a single-neck flask provided with a magnetic stirrer.Dioxane (15 mL) is added to the flask and stirred. A clear solution isobtained. Aqueous solution of sodium hydroxide (10 mL) 0.5 N, is added.The addition of the sodium hydroxide solution results in the mixturebecoming hazy. The mixture is stirred for 16 hours at 25-26° C. Thereaction mixture is centrifuged for 15 minutes. The formation of twophases is observed. The bottom layer is separated and acidified usingdilute aqueous HCl solution to pH=7-7.5 with stirring. Stirring iscontinued for an additional 30 minutes and the pH is determined to bepH=8.0. The crude glatiramer free base solution is filtered.

EXAMPLE 13 Deprotecting Step Conducted at Lower Temperature.

Protected glatiramer acetate, 75 mg, prepared in Example 12 istransferred to a single-neck flask provided with a magnetic stirrer.Tetrahydrofuran (15 mL) is added to the flask and the temperature of thesolution is reduced to 0° C. Aqueous solution of sodium hydroxide (10mL) 0.5 N, is added to the solution while maintaining a temperature of0° C. The addition of the sodium hydroxide results in the solutionbecoming hazy. The solution is stirred for 3 hours at 0° C. Theformation of two phases is observed. The bottom layer is separated andacidified using dilute aqueous HCl solution to pH=7-7.5 with stirring at0° C. Stirring is continued for an additional 30 minutes and the pH isdetermined to be approximately pH=8.0. The crude glatiramer free basesolution is filtered.

EXAMPLE 14

Deprotecting Step with Acetate Buffer.

Protected glatiramer acetate, 75 mg, prepared in Example 12 istransferred to a single-neck flask provided with a magnetic stirrer.Tetrahydrofuran (15 mL) is added to the flask and the temperature of thesolution is reduced to 0° C. Aqueous solution of sodium hydroxide (10mL) 0.5 N, and acetic acid (2 mL) is added to the solution whilemaintaining a temperature of 0° C. and a pH=12. The addition of thesodium hydroxide and acetic acid results in the solution becoming hazy.The solution is stirred for 3 hours at 0° C. The formation of two phasesis observed. The bottom layer is separated and acidified using diluteaqueous HCl solution to pH=7-7.5 with stirring at 0° C. Stirring iscontinued for an additional 30 minutes and the pH is determined to beapproximately pH=8.0. The crude glatiramer free base solution isfiltered.

EXAMPLE 15

Protected glatiramer acetate, 75 mg, prepared in Example 8 istransferred to a single-neck flask provided with a magnetic stirrer.Dioxane (15 mL) is added to the flask and stirred. A clear solution isobtained. Aqueous solution of sodium hydroxide (10 mL) 0.25 N is added.The addition of the sodium hydroxide solution results in the mixturebecoming hazy. The mixture is stirred for 16 hours at 25-26° C. Thereaction mixture is centrifuged for 15 minutes. The formation of twophases is observed. The bottom layer is separated and acidified usingdilute aqueous HCl solution to pH=7-7.5 with stirring. Stirring iscontinued for an additional 30 minutes and the pH is determined to bepH=8.0. The crude glatiramer free base solution is filtered.

EXAMPLE 16 Diafiltration (Tangential Flow Filtration).

The crude glatiramer free base solution prepared in Example 9 is dilutedto 120 with water. The dilute solution is first filtered through a 10 Kdiafiltration membrane, Pellicon XL, PLCCC 10 (50 cm²), available fromMillipore, and then, filtered through a 10 K diafiltration membrane,Pellicon XL, PLCCC 5 (50 cm²), available from Millipore. Theconcentrated solution obtained is lyophilized. A white powder isobtained.

EXAMPLE 17 Chromatographic Method of Purification of Glatirimer Acetate.

The crude glatiramer free base solution prepared in Example 9 issubjected to chromatographic separation. A column for gel filtration,FRACTOGEL TSK HW55 (600×26 mm) is prepared in a Superformance 26 Merckcartridge according to the manufacturer's instructions. The column isequilibrated with 0.2M ammonium acetate buffer pH 5.0, 30 mL ofglatiramer free base solution samples (20 mg/mL, in 0.2 M ammoniumacetate pH 5.0) are loaded on the column and fractions are collectedevery 10 minutes. A fraction having an average molecular weight of 7-8KDa is isolated.

Examples 18-25 relate to a process for preparing glatiramer acetateusing an alkali or alkaline earth metal hydroxide, carbonate, or ahydrogen carbonate.

EXAMPLE 18 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM), N-carboxyanhydrideof L-alanine (620 mg, 5.4 mM), N-carboxyanhydride of γ-benzylL-glutamate (430 mg, 1.6 mM) and N-carboxyanhydride ofN^(ε)-trifluoroacetyl L-lysine (1.01 g, 3.73 mM), are placed in asingle-neck flask (100 mL) with a magnetic stirrer. To this mixture isadded 40 mL of tetrahydrofuran. Diethylamine (10 μL) is added. Theresulting mixture is stirred for 24 hours at a temperature ofapproximately 25° C. The mixture is slowly poured into 100 mL waterwhile stirring. A solid is precipitated. The solid is filtered after 2hours of stirring and washed with water. The solid is resuspended in 100mL water and filtered. The solid is kept under vacuum for approximately12 hours.

EXAMPLE 19 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM), N-carboxyanhydrideof L-alanine (620 mg, 5.4 mM), N-carboxyanhydride of γ-benzylL-glutamate (430 mg, 1.6 mM) and N-carboxyanhydride ofN^(ε)-trifluoroacetyl L-lysine (1.01 g, 3.73 mM), are placed in asingle-neck flask (100 mL) with a magnetic stirrer. To this mixture isadded 40 mL of dioxane. Diethylamine (10 μL) is added. The resultingmixture is stirred for 48 hours at a temperature of approximately 25° C.The mixture is slowly poured into 100 mL water while stirring. A solidis precipitated. The solid is filtered and washed with water. The solidis resuspended in 100 mL water and filtered. The solid is kept undervacuum for approximately 12 hours.

EXAMPLE 20

Cleavage of γ-Benzyl Moiety from the Polypeptide Prepared in Example 2.

The protected polypeptide prepared in Example 19, 100 mg, is suspendedin tetrahydrofuran (20 mL) and cooled in an ice water bath. Concentratedsulfuric acid, 4 mL, is added. The resulting clear solution is stirredfor 20 hours at a temperature of approximately 25° C. The solvent,tetrahydrofuran, is removed by evaporation at 25° C. to form a viscousliquid. Water, 50 mL, is added to the viscous liquid with stirring. Awhite precipitate forms which is filtered under vacuum and dried overphosphorous pentoxide under vacuum at 25° C. for approximately 12 hoursin the dark. A white solid is obtained. The solid is filtered and driedin a dessicator under vacuum. The yield is determined to be 75 mg.

EXAMPLE 21

Cleavage of N^(ε)-trifluoroacetyl Moiety from the Polypeptide Preparedin Example 20.

The protected polypeptide prepared in Example 20, 75 mg, is dispersed in12 mL of tetrahydrofuran, 4 mL of 0.5 M aqueous sodium hydroxide isadded with stirring. The mixture is stirred for 3 hours at ambienttemperature (approximately 22° C.). The lower aqueous layer is separatedand acidified with acetic acid to pH=6.0.

EXAMPLE 22

Cleavage of γ-Benzyl Moiety from the Polypeptide Prepared in Example 19.

The protected polypeptide prepared in Example 19, 1 g, is suspended in50 mL of a mixture prepared of 47 mL concentrated HCl adjusted to 100 mLwith glacial acetic acid. The starting material slowly dissolves forminga slightly turbid solution. The mixture is stirred for 18 hours at atemperature of approximately 22° C. The solution is poured into 1,000 mLof stirred water. A white precipitate is formed. The suspension isstirred for another 3 hours and then filtered. The product is washedwith water and dried under vacuum at 50° C. for approximately 17 hours.

EXAMPLE 23

Cleavage of N^(ε)-trifluoroacetyl Moiety from the Polypeptide Preparedin Example 22.

The protected polypeptide prepared in Example 22, 300 mg, is dispersedin 45 mL of tetrahydrofurane, 25 mL of 0.5 M aqueous sodium hydroxide isadded with stirring. The mixture is stirred for 3 hours at ambienttemperature (approximately 22° C.). A clear, two-phase liquid system isformed. The lower aqueous layer is separated and acidified with aceticacid to pH=6.0. The clear, colorless solution is filled into dialysisbags and dialyzed at ambient temperature once against 0.3% aqueousacetic acid, and then against water until a pH of 5.5 is reached. Thissolution is filtered and lyophilized to yield glatiramer acetate as awhite solid.

EXAMPLE 24 Diafiltration (Tangential Flow Filtration).

The glatiramer acetate solution prepared in Example 21 is adjusted to120 mL with water to provide a 0.5-0.6 mg/mL concentration of theglatiramer acetate. The dilute solution is first filtered through a 30 Kdiafiltration membrane, Pellicon XL, PLCCC 10 (50 cm²), available fromMillipore, and then, filtered through a 3 K diafiltration membrane,Pellicon XL, PLCCC 5 (50 cm²), available from Millipore. Theconcentrated solution obtained is lyophilized to provide glatirameracetate in solid form.

EXAMPLE 25 Chromatographic Method of Purification of Glatiramer Acetate.

The glatiramer as extract of pH=6 prepared in Example 23 is concentratedin vacuo to dryness and subjected to chromatographic separation. Acolumn for gel filtration, FRACTOGEL TSK HW55 (600×26 mm) is prepared ina Superformance 26 Merck cartridge according to the manufacturer'sinstructions. The column is equilibrated with 0.2 M ammonium acetatebuffer pH 5.0, 30 mL of glatiramer free base solution samples (20 mg/mL,in 0.2 M ammonium acetate pH 5.0) are loaded on the column and fractionsare collected. A fraction having an average molecular weight of 7-10 kDais isolated.

Examples 26-33 relate to a process for preparing glatiramer acetateusing an amine or ammonia.

EXAMPLE 26 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM), N-carboxyanhydrideof L-alanine (620 mg, 5.4 mM), N-carboxyanhydride of γ-benzylL-glutamate (430 mg, 1.6 mM) and N-carboxyanhydride ofN^(ε)-trifluoroacetyl L-lysine (1.01 g, 3.73 mM), are placed in asingle-neck flask (100 mL) with a magnetic stirrer. To this mixture isadded 40 mL of tetrahydrofuran. Diethylamine (10 μL) is added. Theresulting mixture is stirred for 24 hours at a temperature ofapproximately 25° C. The mixture is slowly poured into 100 mL waterwhile stirring. A solid is precipitated. The solid is filtered after 2hours of stirring and washed with water. The solid is resuspended in 100mL water and filtered. The solid is kept under vacuum for approximately12 hours.

EXAMPLE 27 Preparation of a Protected Polypeptide.

N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM), N-carboxyanhydrideof L-alanine (620 mg, 5.4 mM), N-carboxyanhydride of γ-benzylL-glutamate (430 mg, 1.6 mM) and N-carboxyanhydride ofN^(ε)-trifluoroacetyl L-lysine (1.01 g, 3.73 mM), are placed in asingle-neck flask (100 mL) with a magnetic stirrer. To this mixture isadded 40 mL of dioxane. Diethylamine (10 μL) is added. The resultingmixture is stirred for 48 hours at a temperature of approximately 25° C.The mixture is slowly poured into 100 mL water while stirring. A solidis precipitated. The solid is filtered and washed with water. The solidis resuspended in 100 mL water and filtered. The solid is kept undervacuum for approximately 12 hours.

EXAMPLE 28

Cleavage of γ-Benzyl Moiety from the Polypeptide Prepared in Example 27.

The protected polypeptide prepared in Example 27, 100 mg, is suspendedin tetrahydrofuran (20 mL) and cooled in an ice water bath. Concentratedsulfuric acid, 4 mL, is added. The resulting clear solution is stirredfor 20 hours at a temperature of approximately 25° C. The solvent,tetrahydrofuran, is removed by evaporation at 25° C. to form a viscousliquid. Water, 50 mL, is added to the viscous liquid with stirring. Awhite precipitate forms which is filtered under vacuum and dried overphosphorous pentoxide under vacuum at 25° C. for approximately 12 hoursin the dark. A white solid is obtained. The solid is filtered and driedin a dessicator under vacuum. The yield is determined to be 75 mg.

EXAMPLE 29

Cleavage of γ-Benzyl Moiety from the Polypeptide Prepared in Example 27.

The protected polypeptide prepared in Example 27, 1 g, is suspended in50 mL of a mixture prepared of 47 mL concentrated HCl adjusted to 100 mLwith glacial acetic acid. The starting material slowly dissolves forminga slightly turbid solution. The mixture is stirred for 18 hours at atemperature of approximately 22° C. The solution is poured into 1,000 mLof stirred water. A white precipitate is formed. The suspension isstirred for another 3 hours and then filtered. The product is washedwith water and dried under vacuum at 50° C. for approximately 17 hours.

EXAMPLE 30

Evaluation of Amines to be Used to Cleave the N^(ε)-trifluoroacetylMoiety from the Polypeptide Prepared in Example 28.

The polypeptide prepared in Example 28, 75 mg, is suspended in 15 mL ofwater. An amine, 7 mL, is added to the suspension to provide an amineconcentration of 3 M. A list of amines is provided in Table IV. Since adeprotected polypeptide is soluble in water, the reaction is monitoredby the clarity of the solution. The results for each of the amines aresummarized in Table IV.

TABLE IV Amine Result Morpholine No clear solution after 48 hours.N-methyl-piperazine No clear solution after 48 hours. DicyclohexylamineNo clear solution after 48 hours. Di-sec-butylamine No clear solutionafter 48 hours. Pyrrolidine No clear solution after 48 hours.Diisopropylamine Clear solution after about 1 hour. Dipropylamine Noclear solution after 48 hours. Isopropylamine Clear solution after about1.5 hours. Methylamine (aqueous) No clear solution after 48 hours.

The results in Table IV clearly show that a free base form of thepolypeptide prepared in Example 28 is formed only upon the addition ofdiisopropylamine or isopropylamine. The results in Table I also showthat dipropylamine, morpholine, N-methyl-piperazine, dicyclohexylamine,di-sec-butylamine, pyrrolidine, and methylamine failed to produce a freebase form of the polypeptide.

Thus, applicants unexpectedly determined that in the second deprotectionstep of the process of the invention, Step (b), diisopropylamine andisopropylamine were the only amines that successfully removed theN^(ε)-trifluoroacetyl group of the lysine moiety.

EXAMPLE 31

Cleavage of the N^(ε)-trifluoroacetyl Moiety from the PolypeptidePrepared in Example 28.

The polypeptide prepared in Example 28, 75 mg, is suspended in 15 mL ofwater. Diisopropylamine, 7 mL, is added to the suspension to provide anamine concentration of 3 M. A milky-white solution becomes clear inapproximately 1 hour and the clear solution is stirred at 25° C. for 20hours. The reaction mixture is evaporated at approximately 25° C. toform crude glatiramer free base in the form of a viscous liquid. Fiftypercent (50%) acetic acid (15 mL) is added to the mixture and stirredfor 30 minutes to form a glatiramer acetate solution.

EXAMPLE 32 Diafiltration (Tangential Flow Filtration).

The glatiramer acetate solution prepared in Example 31 is diluted to 120mL with water. The dilute solution is first filtered through a 30 Kdiafiltration membrane, Pellicon XL, PLCCC 10 (50 cm²), available fromMillipore, and then, filtered through a 3 K diafiltration membrane,Pellicon XL, PLCCC 5 (50 cm²), available from Millipore. Theconcentrated solution obtained is lyophilized to provide glatirameracetate in solid form.

EXAMPLE 33 Chromatographic Method of Purification of Glatiramer Acetate.

The glatiramer acetate solution prepared in Example 31 is concentratedin vacuo to dryness and subjected to chromatographic separation. Acolumn for gel filtration, FRACTOGEL TSK HW55 (600×26 mm) is prepared ina Superformance 26 Merck cartridge according to the manufacturer'sinstructions. The column is equilibrated with 0.2 M ammonium acetatebuffer pH 5.0, 30 mL of glatiramer free base solution samples (20 mg/mL,in 0.2 M ammonium acetate pH 5.0) are loaded on the column and fractionsare collected. A fraction having an average molecular weight of 7-10 kDais isolated.

While the invention has been described with particular reference tocertain embodiments thereof, it will be understood that changes andmodifications may be made by those of ordinary skill within the scopeand spirit of the following claims:

1-31. (canceled)
 32. A process for preparing a polypeptide comprisingL-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceuticallyacceptable salt thereof, wherein said process comprises treating aprotected polypeptide with an aqueous solution of an alkali or alkalineearth metal hydroxide to form a polypeptide or a pharmaceuticallyacceptable salt thereof.
 33. A process for preparing a polypeptidecomprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or apharmaceutically acceptable salt thereof, wherein said processcomprises: (a) polymerizing a mixture of N-carboxyanhydride ofL-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of aprotected L-glutamate and N-carboxyanhydride of a protected L-lysine, ina polar aprotic solvent in the presence of an initiator, to form aprotected polypeptide; and (b) adding an aqueous solution of an alkalior alkaline earth metal hydroxide to the protected polypeptide formed inStep (a) to form a polypeptide or a pharmaceutically acceptable saltthereof.
 34. A process for preparing glatiramer acetate comprising: (a)polymerizing a mixture of N-carboxyanhydride of L-tyrosine,N-carboxyanhydride of L-alanine, N-carboxyanhydride of a γ-benzylL-glutamate and N-carboxyanhydride of N^(ε)-trifluoroacetyl L-lysine, ina polar aprotic solvent in the presence of an initiator, to form aprotected glatiramer; (b) adding an aqueous solution of an alkali oralkaline earth metal hydroxide to the protected glatiramer formed inStep (a) to form a glatiramer; and (c) treating the glatiramer withacetic acid to form glatiramer acetate.
 35. The process according toclaim 33, wherein Step (b) is conducted at a temperature of from about−78° C. to about 40° C.
 36. The process according to claim 35, whereinStep (b), is conducted at a temperature of from about −25° C. to about30° C.
 37. The process according to claim 36, wherein Step (b), isconducted at a temperature of from about −10° C. to about 10° C.
 38. Theprocess according to claim 37, wherein Step (b), is conducted at atemperature of from about 0° C.
 39. The process according to claim 32wherein the pH is from about 13 to about
 14. 40. The process accordingto claim 32 which additionally comprises a buffer.
 41. The processaccording to claim 40, wherein the buffer is an acetate buffer and thepH is from about 8 to about
 12. 42. The process according to claim 32,wherein the alkali or alkaline earth metal hydroxide is selected fromthe group consisting of calcium hydroxide, lithium hydroxide, magnesiumhydroxide, potassium hydroxide, sodium hydroxide and mixtures thereof.43. The process according to claim 42, wherein the alkali or alkalineearth metal hydroxide is sodium hydroxide.
 44. The process according toclaim 32, wherein the alkali or alkaline earth metal hydroxide ispresent in an amount of from about 0.1 wt. % to about 400 wt. %, basedon the total weight of the polypeptide or pharmaceutically acceptablesalt thereof.
 45. The process according to claim 44, wherein the alkalior alkaline earth metal hydroxide is present in an amount of from about10 wt. % to about 300 wt. %, based on the total weight of thepolypeptide or pharmaceutically acceptable salt thereof.
 46. The processaccording to claim 45, wherein the alkali or alkaline earth metalhydroxide is present in an amount of from about 140 wt. % to about 260wt. %, based on the total weight of the polypeptide or pharmaceuticallyacceptable salt thereof.
 47. A polypeptide or a pharmaceuticallyacceptable salt thereof which is prepared by a process comprisingtreating a protected polypeptide with an aqueous solution of an alkalior alkaline earth metal hydroxide to form the polypeptide orpharmaceutically acceptable salt thereof, wherein said polypeptidecomprises L-tyrosine, L-alanine, L-glutamate and L-lysine.
 48. Apolypeptide or a pharmaceutically acceptable salt thereof which isprepared by a process comprising: (a) polymerizing a mixture ofN-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine,N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride ofa protected L-lysine, in a polar aprotic solvent in the presence of aninitiator, to form a protected polypeptide; and (b) adding an aqueoussolution of an alkali or alkaline earth metal hydroxide to the protectedpolypeptide formed in Step (a) to form a polypeptide or apharmaceutically acceptable salt thereof.
 49. Glatiramer acetate whichis prepared by a process comprising (a) polymerizing a mixture ofN-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine,N-carboxyanhydride of a γ-benzyl L-glutamate and N-carboxyanhydride ofN^(ε)-trifluoroacetyl L-lysine, in a polar aprotic solvent in thepresence of an initiator, to form a protected glatiramer; (b) adding anaqueous solution of an alkali or alkaline earth metal hydroxide to theprotected glatiramer formed in Step (a) to form a glatiramer; and (c)treating the glatiramer formed in Step (b) with acetic acid to formglatiramer acetate. 50-77. (canceled)